Sindbis control virus

ABSTRACT

Disclosed are compositions and methods related to replication deficient Sindbis viruses that are able to function as controls for nucleic acid diagnostic assays (e.g., nucleic acid sequencing based assays and/or nucleic acid amplification based assays).

PRIORITY CLAIM

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/182,104, filed Jun. 19, 2015, which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 31, 2016, isnamed SCX_00325_SL.txt and is 144,758 bytes in size.

BACKGROUND

Regulatory agencies, such as the FDA, CLIA and CAP, generally requiredevelopers of nucleic acid-based in vitro diagnostic devices forpathogen detection to include quality controls in their regulatorysubmissions. Such quality control materials are important tools for thedetection of analytical errors, the monitoring of long-term performanceof diagnostic test kits, and the identification of changes in random orsystematic error. A well-designed laboratory quality control programwill generally incorporate at least some form of control that providesadded confidence in the reliability of results obtained for unknownspecimens.

Whole process controls are needed to monitor the entire analyticalprocess, including sample lysis, nucleic acid extraction, amplification,detection and interpretation of results. Such controls can be naturalmaterial derived from infected patients, which have the advantage ofbehaving very similarly to a clinical sample. However, such naturalsource controls often have limited and unpredictable availability,concentration and stability. The use of cultured virus to generatepositive controls alleviates some of these problems, but virus cultureis often unavailable or technologically difficult. In addition, thepreparation of large amount of human pathogens caries significant safetyrisks and is expensive.

Positive controls for amplification and detection are often provided aspart of diagnostic test kits. The materials often have a known amount ofinput copy number and verify the integrity of the reaction componentsand instrument. However, such controls are not usually taken through thesample lysis or nucleic acid extraction process and are therefore unableto detect errors arising from these steps. Examples of this type ofcontrol include a non-infectious DNA plasmid containing the targetsequence, purified RNA transcripts, or packaged RNA materials such asArmored RNA. These materials often also suffer from their limitedstability at ambient temperatures.

Internal controls contain a non-target nucleotide sequence that isco-extracted and co-amplified with the target nucleic acid. Internalcontrols confirm the integrity of the reagents (e.g., polymerase,primers, etc.), equipment function (e.g., thermal cycler), and theabsence of inhibitors in the sample. The internal control can take theform of a non-target organism that is added to the sample prior tosample lysis and extraction. Alternatively, it could be anon-infectious, non-target DNA or RNA sequence that is added to thesample either prior to or after sample lysis and extraction.

Thus, there is a need for improved compositions able to serve ascontrols in diagnostic assays.

SUMMARY

Provided herein are compositions and methods related to replicationdeficient Sindbis viruses that are able to function as controls fornucleic acid diagnostic assays (e.g., nucleic acid sequencing basedassays and/or nucleic acid amplification based assays).

In certain aspects, disclosed herein is a replication deficientrecombinant Sindbis virus comprising a RNA genome comprising (a) an openreading frame (ORF) encoding functional Sindbis non-structural proteinsand (b) a heterologous (i.e., non-Sindbis) RNA sequence. In someembodiments, the ORF encoding the functional Sindbis non-structuralproteins is located 5′ of the heterologous RNA sequence.

In some embodiments, the ORF encoding the Sindbis non-structuralproteins encodes a nsP1 protein, a nsP2 protein, a nsP3 protein and ansP4 protein. In some embodiments, the ORF encoding Sindbisnon-structural proteins has a nucleotide sequence that is at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to nucleotides 1-7648 of SEQ ID NO: 1. Thesenucleotides encode non-structural Sindbis proteins.

In some embodiments, the RNA genome of the replication deficient Sindbisvirus lacks a sequence encoding a functional version of one or more ofthe Sindbis structural proteins (e.g., Sindbis capsid protein, E3protein, E2 protein, 6k protein and/or E1 protein),In some embodiments,the RNA genome lacks an RNA sequence encoding any functional Sindbisstructural proteins. In some embodiments, the heterologous RNA sequencereplaces the ORF encoding the Sindbis structural proteins in the RNAgenome.

In some embodiments, the replication deficient recombinant Sindbis virusof claim any one of claims 1 to 8, wherein the RNA genome comprises a26S subgenomic promoter at the 3′ end of the ORF encoding the Sindbisnon-structural proteins.

In some embodiments, the heterologous RNA sequence in the RNA genomecomprises a non-Sindbis RNA virus sequence or a retrovirus sequence. Insome embodiments, the heterologous RNA sequence includes at least 10,50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900 or 2000 bp of a non-Sindbis RNA virus sequence or aretrovirus sequence. In some embodiments, the heterologous RNA sequenceincludes 100-300 bp of a non-Sindbis RNA virus sequence or a retrovirussequence. In some embodiments, the heterologous RNA sequence includes100-200 bp of a non-Sindbis RNA virus sequence or a retrovirus sequence.In some embodiments, the heterologous RNA sequence is at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to a non-Sindbis RNA virus sequence or aretrovirus sequence. In some embodiments, the non-Sindbis RNA virussequence or retrovirus sequence comprises one or more mutations thatconvey a drug resistant phenotype when present in the non-Sindbis RNAvirus or the retrovirus. For example, in some embodiments thenon-Sindbis RNA virus sequence or retrovirus sequence comprises at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations that convey a drugresistant phenotype when present in the non-Sindbis RNA virus or theretrovirus.

In some embodiments, the heterologous RNA sequence comprises anon-Sindbis RNA virus sequence. In some embodiments, the non-Sindbis RNAvirus sequence is an Ebolavirus sequence, an influenza virus sequence, aSARS virus sequence, a hepatitis C virus sequence, a West Nile virussequence, a Zika virus sequence, a poliovirus sequence or a measlesvirus sequence.

In some embodiments, the non-Sindbis RNA virus sequence is an Ebolavirussequence (e.g., a Zaire ebolavirus sequence, a Bundibugyo ebolavirussequence, a Reston ebolavirus sequence, a Sudan ebolavirus sequence or aTai Forest ebolavirus sequence). In some embodiments, the Ebolavirussequence comprises at least a portion of an Ebolavirus GP gene sequence,an Ebolavirus NP gene sequence or an Ebolavirus VP24 gene sequence. Insome embodiments, the heterologous RNA sequence does not encode afunctional Ebola protein (e.g., the heterologous RNA sequence encodestruncated Ebola proteins, Ebola proteins with frame-shift mutationsand/or Ebola protein sequences lacking a start codon). In someembodiments, the heterologous RNA sequence comprises a sequence at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3. SEQID NO: 2 is the nucleotide sequence of a GP Ebola target sequence usedin an exemplary Ebola Sindbis control virus described in Example 1. SEQID NO: 3 is the nucleotide sequence of a NP/VP24 Ebola target sequenceused in an exemplary Ebola Sindbis control virus described in Example 1.The portion of the Ebola NP gene consists of nucleotides 1 to 1577 ofSEQ ID NO: 3, the portion of the Ebola VP24 gene consists of nucleotides1578 to 2127 and the sequence of the human RNAse P internal controlconsists of nucleotides 2128 to 2217.

In some-embodiments, the heterologous RNA sequence comprises aretrovirus sequence. In some embodiments, the retrovirus sequence is anHIV-1 sequence, an HIV-2 sequence, an HTLV-1 sequence, or an HTLV-IIsequence.

In some embodiments, the heterologous RNA sequence comprises an HIV-1sequence. In some embodiments, the HIV-1 sequence comprises one or moremutations that, when present in a HIV-1 virus, conveys a drug resistancephenotype (e.g., resistance to a protease inhibitor, a nucleosideanalogue reverse transcriptase inhibitor and/or a non-nucleoside analogreverse transcriptase inhibitor). For example, in some embodiments theHIV-1 virus sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 mutations that convey a drug resistant phenotype. In someembodiments, the one or more mutations, when present in HIV-1 virus,convey resistance to a drug selected from the group consisting of:atazanavir, ritonavir, darunavir, fosamprenavir, indinavir, lopinavir,nelfinavir, saquinavir, tipranavir, abacavir, didanosine, emtricitabine,lamivudine, stavudine, tenofovir, zidovudine, efavirenz, etavirine,nevirapine or rilpivirine. In some embodiments, the one or moremutations are selected from the group consisting of L24I, D30N, V32I,M46I, I47V, G48V, 150V, I54M,

G73S, L76V, V82A, I84V, N88D, L90M, M41L, K65R, D67N, T69S insert SS,K7OR, L74V, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y, K219Q,L100I, K101E, K103N, V106A, V1081, Y181C, Y188L, G190A, P225H and M230L.In some embodiments, the one or more mutations are selected from thegroup consisting of L24I (TTA to ATA), D30N (GAT to AAT), V32I (GTA toATA), M46I (ATG to ATA), I47V (ATA to CTA), G48V (GGG to GTG), I50V (ATTto GTT), I54M (ATC to ATG), G73 S(GGT to GCT), L76V (TTA to GTA), V82A(GTC to GCC), I84V (ATA to GTA), N88D (AAT to GAT), L9OM (TTG to ATG),M41L (ATG to TTG), K65R (AAA to AGA), D67N (GAC to AAC), T69S insert SS(ACT to TCT and insertion of TCC and TCC), K7OR (AAA to AGA), L74V (TTAto GTA), F77L (TTC to CTC), Y115F (TAT to TTT), F116Y (TTT to TAT),Q151M (CAG to ATG), M184V (ATG to GTG), L210W (TTG to TGG), T215Y (ACCto TAC), K219Q (AAA to CAA), L100I (TTA to ATA), K101E (AAA to GAA),K103N (AAA to AAC), V106A (GTA to GCA), V108I (GTA to ATA), Y181C (TATto TGT), Y188L (TAT to TTA), G190A (GGA to GCA), P225H (CCT to CAT) andM230L (CCT to CAT). In some embodiments, the HIV-1 sequence comprises atleast a portion of an HIV-1 gene selected from p7, pl, p6, HIV protease,reverse transcriptase, p51 RNAse, integrase and gp120. In someembodiments, the HIV-1 sequence comprises at least a portion of p7, pl,p6, HIV protease, reverse transcriptase and integrase. In certainembodiments, the HIV-1 sequence comprises at least a portion of 6p120,wherein the portion comprises the V1-V5 variable loops. In someembodiments, the HIV-1 sequence comprises a sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to nucleotides 1900 through 5400and/or 6300 through 7825 of the HXB2 strain of HIV-1 (SEQ ID NO: 4). SEQID NO: 4 is the nucleotide sequence of the HIV-1 HXB2 genome. In someembodiments, the HIV-1 sequence is identical to nucleotides 1900 through5400 and/or 6300 through 7825of the HXB2 strain of HIV-1 (SEQ ID NO: 4)except for the presence of the mutations that convey a drug resistancephenotype.

In some embodiments, the heterologous RNA sequence comprises a sequenceat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical to SEQ ID NO: 5 and/or SEQ IDNO: 7. SEQ ID NO: 5 is the nucleotide sequence of a 5′ multi-mutantHIV-1 target sequence comprising a number of drug resistance mutations,used in an exemplary multi-mutant HIV-1 control virus described inExample 2. SEQ ID NO: 7 is the nucleotide sequence of a 3′ mutant HIV-1target sequence used in an exemplary multi-mutant HIV-1 control virusdescribed in Example 2.

In some embodiments, the heterologous RNA sequence comprises a sequenceat least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical to SEQ ID NO: 6 and/or SEQ IDNO: 8. SEQ ID NO: 6 is the nucleotide sequence of a 5′ wild-type HIV-1target sequence in an exemplary HIV-1 control virus, described inExample 2. SEQ ID NO: 8 is the nucleotide sequence of a 3′ wild-typeHIV-1 target sequence used in an exemplary HIV-1 control virus,described in Example 2.

In some embodiments, the heterologous RNA sequence comprises a sequencethat is at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical to either nucleotides1-3446, nucleotides 3294-5575, nucleotides 5425-7722, or nucleotides7542-10272 of SEQ ID NO: 15.

In some embodiments, the RNA genome of the replication deficient Sindbisvirus comprises a nucleotide sequence that is at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

In certain aspects, provided herein is a composition comprising areplication deficient Sindbis virus described herein. In certainaspects, the composition comprises two or more of the replicationdeficient Sindbis viruses described herein. For example, in someembodiments, the composition comprises a replication deficient Sindbisvirus comprising a RNA genome comprising a sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to SEQ ID NO: 11 and a replicationdeficient Sindbis virus comprising a RNA genome comprising a sequencethat is at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 13. Insome embodiments, the composition comprises a replication deficientSindbis virus comprising a RNA genome comprising a sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical to SEQ ID NO: 12 and areplication deficient Sindbis virus comprising a RNA genome comprising asequence that is at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:14. In some embodiments, the composition further comprises human DNA. Insome embodiments, the replication deficient Sindbis virus is in a humanbodily fluid. In some embodiments, the human bodily fluid is humanplasma (e.g., defibrinated human plasma). In some embodiments, thecomposition further comprises a preservative, such as sodium azide.

In some embodiments, the composition comprises a replication deficientSindbis virus comprising a RNA genome comprising a sequence that is atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% identical to either nucleotides 1-3446,nucleotides 3294-5575, nucleotides 5425-7722, or nucleotides 7542-10272of SEQ ID NO: 15.

In certain aspects, provided herein is a nucleic acid molecule encodingthe RNA genome of the replication deficient Sindbis virus describedherein. In some embodiments, the nucleic acid molecule is a DNAmolecule. In some embodiments, the nucleic acid molecule is an RNAmolecule. In some embodiments, the nucleic acid molecule is a plasmid(e.g., a circular plasmid or a linearized plasmid, such as a circularexpression plasmid or a linearized expression plasmid). In someembodiments, the nucleic acid molecule is isolated. In certainembodiments, provided herein is a cell comprising a nucleic aciddescribed herein. In some embodiments ,the cell is a BHK cell.

In certain aspects, provided herein is a method of making a replicationdeficient Sindbis virus. In certain embodiments, the method includes thestep of transfecting a cell (e.g., a BHK cell) with a nucleic acidmolecule (e.g., an RNA molecule) encoding the RNA genome of areplication deficient Sindbis virus described herein and with a nucleicacid (e.g., an RNA molecule) encoding functional Sindbis structuralproteins. In some embodiments, the cell is then cultured underconditions such that the cell produces the replication deficient Sindbisvirus into the culture medium. In some embodiments, the method furthercomprises collecting the replication deficient Sindbis virus (e.g., bycollecting the culture supernatant). In some embodiments, the methodfurther comprises filtering and/or heat inactivating the culturesupernatant. In some embodiments, the method further comprisesdetermining the titer of the virus (e.g., using real-time PCR).

In certain aspects, provided herein are methods of testing a diagnosticassay by running the diagnostic assay on a composition comprising thereplication deficient Sindbis virus described herein. In someembodiments, the diagnostic assay is a nucleic acid amplification baseddiagnostic assay. In some embodiments, the diagnostic assay is asequencing based diagnostic assay. In some embodiments the diagnosticassay is an assay for the detection of a RNA virus and/or a retrovirus.In some embodiments, the diagnostic assay is an assay for the detectionof Ebolavirus, an influenza virus, a SARS virus, a hepatitis C virus, aWest Nile virus, a Zika virus, a poliovirus, a measles virus, an HIV-1virus, an HIV-2 virus, an HTLV-I virus and/or an HTLV-II virus. Incertain embodiments, the heterologous RNA sequence in the RNA genome ofthe replication deficient Sindbis virus contains the target sequencedetected in the diagnostic assay. In some embodiments, the methodincludes the performance of a sample lysis step on the compositioncomprising the replication deficient Sindbis virus. In some embodiments,the method comprises performing a nucleic acid extraction step. In someembodiments, the method comprises performing a nucleic acidamplification step (e.g., performing a real-time nucleic acidamplification/detection process). In some embodiments, the methodcomprises performing a nucleic acid sequencing step. In some embodimentsthe method comprises performing a nucleic acid detection step.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a schematic depiction of the genomic organization ofSindbis virus. Some of the genes shown encode nonstructural proteins(nsP1-4), which include the helicase and RNA polymerase. Some of thegenes are the structural genes, and encode the capsid (C) as well asproteins involved in budding.

FIG. 2 shows a schematic depiction of the genomic organization of aSindbis control vector of certain embodiments described herein.

FIG. 3 shows an exemplary schematic for the production of recombinantSindbis control viruses.

FIG. 4 shows the results of a TaqMan real time quantitation assay ofSindbis control samples unstressed (time=0) or stressed for 1, 3, 5, 11or 22 days at 37° C.

FIG. 5 shows the results of a TaqMan real time quantitation assay ofnon-stressed Sindbis control samples or samples stressed through one,two or three Freeze/Thaw cycles.

FIG. 6 shows the results of a TaqMan real time quantitation assay of aSindbis control virus stored frozen at −20° C., stored refrigerated at2-8° C. or stored at ambient lab temperature across seven months.

FIG. 7 shows a workflow overview for the production of Sindbis controlvirus.

FIG. 8 shows a map of the SinRep SC vector. Figure discloses “His8” asSEQ ID NO: 16.

FIG. 9 consists of two maps of the Zika virus genome. The genome wasdivided into four regions for the construction of four different Zikavirus reference materials, and each region is depicted by a rectangle. Afirst reference material comprises nucleotides 1 to 3446 of the Zikavirus from GenBank Accession number EU545988.1, referred to as the “ZikaEnv Construct” (Construct -1). A second reference material comprisesnucleotides 3294 to 5575, referred to as the “Zika NS2/NS3 Construct”(Construct -2). A third reference material comprises nucleotides 5425 to7722, referred to as the “Zika NS4 Construct” (Construct -3). A fourthreference material comprises nucleotides 7542 to 10272, referred to asthe “Zika NS5 Construct” (Construct -4).

FIG. 10A depicts nucleotides 1 to 3446 of the Zika virus from GenBankAccession number EU545988.1, referred to as the “Zika Env Construct,”which includes the NS1 gene. This portion of the Zika virus genome wasintegrated into a Zika virus reference material.

FIG. 10B depicts of nucleotides 3294 to 5575 of the Zika virus fromGenBank Accession number EU545988.1, referred to as the “Zika NS2/NS3Construct,” which includes the NS2 and NS3 genes as well as a portion ofthe NS1 gene. This portion of the Zika virus genome was integrated intoa Zika virus reference material.

FIG. 10C depicts nucleotides 5425 to 7722 of the Zika virus from GenBankAccession number EU545988.1, referred to as the “Zika NS4 Construct,”which includes the NS4A and NS4B genes as well as a portion of the NS3gene. This portion of the Zika virus genome was integrated into a Zikavirus reference material.

FIG. 10D depicts nucleotides 7542 to 10272 of the Zika virus fromGenBank Accession number EU545988.1, referred to as the “Zika NS5Construct,” which includes the NS5 gene and a portion of the NS4B gene.This portion of the Zika virus genome was integrated into a Zika virusreference material.

FIG. 11A shows stability results of TaqMan real time quantitation of anH7N9 influenza reference material stored at −20° C., 4° C., or roomtemperature(˜25°) for seventeen months. The results depicted are forreference materials formulated with buffer.

FIG. 11B shows stability results of TaqMan real time quantitation of anH7N9 influenza reference material stored at −20° C., 4° C., or roomtemperature (˜25°) for seventeen months. The results depicted are forreference materials formulated with human plasma.

FIG. 12 shows stability results of TaqMan real time quantitation of aH7N9 influenza reference material stored at ambient temperature forseventeen months. Each error bar corresponds to 1 standard deviationfrom the mean.

DETAILED DESCRIPTION General

Provided herein are compositions and methods related to replicationdeficient

Sindbis viruses that are able to function as controls for nucleic aciddiagnostic assays (e.g., nucleic acid sequencing based assays and/ornucleic acid amplification based assays). In certain aspects, providedherein are Sindbis control virus are useful as whole process controls,positive controls and/or internal controls in nucleic acid diagnosticassays. Such control virus can benefit diagnostics manufacturers byproviding a less expensive, consistent and safe source of startingmaterial for controls. The control virus described herein use Sindbisvirus, an RNA containing enveloped virus which can be engineered tocontain target RNA sequences such as sequences from another virus and/oran internal control sequence. The Sindbis virus coat provides the RNAgenome with improved stability. In some embodiments, the recombinantSindbis virus system described herein results in viral particles thatare packaged, so they can be used to evaluate nucleic acid extractionprocesses that are used before nucleic acid detection. Also providedherein are compositions comprising such viruses, nucleic acid moleculesencoding the RNA genome of such control viruses, methods of making suchcontrol viruses and methods of using such control viruses.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “biological sample,” “tissue sample,” or simply “sample” eachrefers to a collection of cells obtained from a tissue of a subject. Thesource of the tissue sample may be solid tissue, as from a fresh, frozenand/or preserved organ, tissue sample, biopsy, or aspirate; blood or anyblood constituents, serum, blood; bodily fluids such as cerebral spinalfluid, amniotic fluid, peritoneal fluid or interstitial fluid, urine,saliva, stool, tears; or cells from any time in gestation or developmentof the subject.

The term “control” includes any portion of an experimental systemdesigned to demonstrate that the factor being tested is responsible forthe observed effect, and is therefore useful to isolate and quantify theeffect of one variable on a system.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. The term “gene” applies to a specificgenomic sequence, as well as to a cDNA or an mRNA encoded by thatgenomic sequence.

As used herein, the term “heterologous RNA” refers to RNA present in arecombinant Sindbis virus that is not derived from wild-type Sindbisvirus. For example, heterologous RNA in a Sindbis virus can be an RNAsequence normally found in a different virus (e.g., a different RNAvirus or retrovirus), can be an RNA sequence normally found a non-viralorganism, or can be a completely artificial RNA sequence.

The term “isolated nucleic acid” refers to a polynucleotide of naturalor synthetic origin or some combination thereof, which (1) is notassociated with the cell in which the “isolated nucleic acid” is foundin nature, and/or (2) is operably linked to a polynucleotide to which itis not linked in nature.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleotide sequences provided herein, U nucleotides are interchangeablewith T nucleotides.

As used herein, the term “Sindbis virus” includes viral particles madeup of an icosahedral capsid that comprises Sindbis virus capsid, E1 andE2 proteins encompassing a single-stranded RNA genome. The RNA genomecan include non-Sindbis RNA (i.e., heterologous RNA) and does not needto include all parts of the wild-type Sindbis genome. For example, insome embodiments the RNA genome does not encode one or more of theSindbis structural proteins.

Replication Deficient Sindbis Control Viruses

In certain embodiments, provided herein are replication deficientSindbis control viruses. In some embodiments, such viruses have an RNAgenome that includes (a) an open reading frame (ORF) encoding functionalSindbis non-structural proteins and (b) a heterologous (i.e.,non-Sindbis) RNA sequence. In some embodiments, the ORF encoding thefunctional Sindbis non-structural proteins is located 5′ of theheterologous RNA sequence. In some embodiments, the heterologous RNAsequence is a sequence from a different RNA virus (e.g., an Ebolavirussequence, an influenza virus sequence, a SARS virus sequence, ahepatitis C virus sequence, a West Nile virus sequence, a Zika virussequence, a poliovirus sequence or a measles virus sequence) or asequence from a retrovirus (e.g., an HIV-1 sequence, an HIV-2 sequence,an HTLV-1 sequence, or an HTLV-II sequence).

Wild-type Sindbis virus is a member of Alphavirus genus, familyTogaviridae. The viral genome is approximately 11,700 nucleotides. Assuch, Sindbis virus has approximately the same genomic complexity asmany human pathogenic viruses, including, for example, HIV-1 (9270nucleotides), HCV (9700 nucleotides) and Ebola Zaire (18959nucleotides). This offers a technical advantage over certain othertechnologies used to package RNA controls, such as Armored RNA, whichare based on MS2 bacteriophage technology and produce recombinant RNAmolecules as small as 900 bases in length, which in many instances doesadequately reflect the complexity or RNA secondary structure of thepathogenic viruses found in patient samples.

As depicted in FIG. 1, wild-type Sindbis virus contains asingle-stranded positive sense genomic RNA which encodes both viralstructural proteins (for capsid assembly and viral budding) as well asthe nonstructural proteins (such as the replication enzymes). Upon entryof the virus into a cell, the RNA is released into cytoplasm and drivesproduction of the viral replicase proteins (non-structural proteins1-4). These proteins form replication and transcription complexes andare responsible for generating the negative strand of the genomic RNA.Promoters in the negative strand genomic RNA drive transcription of twomRNA species: The full-length genomic RNA encodes the nonstructuralproteins and the smaller subgenomic RNA encodes the structural proteins.The 5′ ends of both transcripts are capped with 7-methylguanosine andthe 3′ ends are polyadenylated.

In certain embodiments, the recombinant Sindbis control virusesdescribed herein are replication deficient. In some embodiments, anymethod can be used to render the recombinant Sindbis control virusreplication deficient. For example, in some embodiments the Sindbiscontrol virus does not encode one or more functional structuralproteins. For example, in some embodiments, the In some embodiments therecombinant Sindbis control virus genome does not encode one or morefunctional nonstructural proteins. In some embodiments, the Sindbiscontrol virus does not encode a functional nsP1 protein, a functionalnsP2 protein, a functional nsP3 protein and/or a functional nsP4protein.

As described herein, separation of the Sindbis viral genome into two ORFfacilitates the manipulation of the viral genome through replacement ofthe genes coding for the structural proteins with target sequences. Thismodified genomic RNA can be transcribed in vitro and introduced intocells along with a helper RNA (e.g., encoding structural proteins notencoded for in the modified RNA genome) for the defective virus. In someembodiments, the helper RNA encodes the four structural proteinsrequired for Sindbis Virus packaging. In some embodiments, the helperRNA does not contain a packaging signal, and so does not getincorporated into the assembled viral particles. Thus, in certainembodiments, the viral particles produced therefore contain the targetsequences but are replication defective because they do not bear thegenetic information to produce the structural proteins. The recombinantviruses produced are effective quality control materials since they bearthe selected target sequences, but the design of the recombinant Sindbissystem provided herein ensures that the virus particles are safe and arenot capable of establishing continuous infection. This is a distinctadvantage for these materials over patient sourced or cultured viralmaterials as controls. FIG. 2 illustrates transcribed RNAs used forassembly of replication defective recombinant viruses.

Assembly of the virus particle occurs at the plasma membrane. Aheterodimer of the structural proteins, E1 and E2, inserts into theplasma membrane and the E2 cytoplasmic tail is thought to provide thebinding site for the nucleocapsid. This interaction between E2 and thenucleocapsid is thought to initiate the actual budding and release ofthe virus. When recombinant Sindbis viruses are produced in culturedcells, the virus particles are collected from the culture media, wherethey typically reach concentrations greater than 1×10⁸ viral copies/mL.The budding process results in the recombinant Sindbis virus beingenveloped into a lipid bilayer. This is important since the structure ofthe recombinant virus is thus similar to many other viruses generallyclassified as RNA-containing enveloped viruses such as HIV-1, HCV, HTLV,Influenza, and SARS. Therefore, the replication deficient Sindbisvectors described herein can be a true whole process control as theyundergo sample lysis and nucleic acid processing similar to humanpathogenic viruses that may be found in patient samples.

In recombinant Sindbis viruses, the target sequences replace thestructural genes. This gives the system great flexibility in the size ofthe target sequences that can be accommodated and packaged efficiently.Target sequences of less than 100 bp to greater than 4000 bp can beefficiently incorporated in the recombinant viruses. The ability toaccommodate large sequences is a distinct advantage, especially whenproducing controls for multiplexed assays. Multiple target sequences(from different pathogens or from different genes within the samepathogen) can be combined in one recombinant virus to form a multiplexcontrol.

In some embodiments, the Sindbis control viruses described hereincomprise HIV-1 sequence and are therefore useful as a control for HIV-1diagnostic assays. In some embodiments, the HIV-1 sequence in theSindbis control virus is distinct from naturally occurring HIV-1 virussequence in that it contains resistance mutations arising from multipleclasses of current HIV-1 therapies. Such multiplexed mutations do notoccur in nature. In some embodiments, the control virus has the variousdrug resistance mutations present at the same allelic ratio. Thisprovides users with a clear expectation for their test results. Incertain embodiments, stop codons are engineered into the HIV-1 sequencesso that no functional HIV-1 proteins are produced.

In some aspects provided herein is an HIV-1 Sindbis control virus thatcomprises an HIV-1 sequence in its RNA genome. In some embodiments, theHIV-1 sequence comprises one or more mutations that, when present in aHIV-1 virus, conveys a drug resistance phenotype (e.g., resistance to aprotease inhibitor, a nucleoside analogue reverse transcriptaseinhibitor and/or a non-nucleoside analog reverse transcriptaseinhibitor). For example, in some embodiments the HIV-1 virus sequencecomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mutations thatconvey a drug resistant phenotype. In some embodiments, the one or moremutations, when present in HIV-1 virus, convey resistance to a drugselected from the group consisting of: atazanavir, ritonavir, darunavir,fosamprenavir, indinavir, lopinavir, nelfinavir, saquinavir, tipranavir,abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir,zidovudine, efavirenz, etavirine, nevirapine or rilpivirine. In someembodiments, the one or more mutations are selected from the groupconsisting of L241, D30N, V321, M461, I47V, G48V, 150V, I54M, G73S,L76V, V82A, I84V, N88D, L90M, M41L, K65R, D67N, T69S insert SS, K7OR,L74V, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y, K219Q, L100I,K101E, K103N, V106A, V1081, Y181C, Y188L, G190A, P225H and M230L. Insome embodiments, the one or more mutations are selected from the groupconsisting of L241 (TTA to ATA), D30N (GAT to AAT), V321 (GTA to ATA),M461 (ATG to ATA), I47V (ATA to CTA), G48V (GGG to GTG), 150V (ATT toGTT), I54M (ATC to ATG), G73S(GGT to GCT), L76V (TTA to GTA), V82A (GTCto GCC), I84V (ATA to GTA), N88D (AAT to GAT), L9OM (TTG to ATG), M41L(ATG to TTG), K65R (AAA to AGA), D67N (GAC to AAC), T69S insert SS (ACTto TCT and insertion of TCC and TCC), K7OR (AAA to AGA), L74V (TTA toGTA), F77L (TTC to CTC), Y115F (TAT to TTT), F116Y (TTT to TAT), Q151M(CAG to ATG), M184V (ATG to GTG), L210W (TTG to TGG), T215Y (ACC toTAC), K219Q (AAA to CAA), L100I (TTA to ATA), K101E (AAA to GAA), K103N(AAA to AAC), V106A (GTA to GCA), V108I (GTA to ATA), Y181C (TAT toTGT), Y188L (TAT to TTA), G190A (GGA to GCA), P225H (CCT to CAT) andM230L (CCT to CAT). In some embodiments, the HIV-1 sequence comprises atleast a portion of an HIV-1 gene selected from p7, pl, p6, HIV protease,reverse transcriptase, p51 RNAse, integrase and gp120. In someembodiments, the HIV-1 sequence comprises at least a portion of p7, pl,p6, HIV protease, reverse transcriptase and integrase. In certainembodiments, the HIV-1 sequence comprises at least a portion of 6p120,wherein the portion comprises the V1-V5 variable loops. In someembodiments, the HIV-1 sequence comprises a sequence that is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to nucleotides 1900 through 5400and/or 6300 through 7825 of the HXB2 strain of HIV-1 (SEQ ID NO: 4). Insome embodiments, the HIV-1 sequence is identical to nucleotides 1900through 5400 and/or 6300 through 7825of the HXB2 strain of HIV-1 (SEQ IDNO: 4) except for the presence of the mutations that convey a drugresistance phenotype. In some embodiments, the heterologous RNA sequencecomprises a sequence at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQID NO: 5 and/or SEQ ID NO: 7. In some embodiments, the heterologous RNAsequence comprises a sequence at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identical toSEQ ID NO: 6 and/or SEQ ID NO: 8.

In some embodiments, the Sindbis control viruses described hereincomprise Ebolavirus sequence and are therefore useful as a control forEbolavirus diagnostic assays. In some embodiments, the Ebolavirussequence comprises at least a portion of an Ebolavirus GP gene sequence,an Ebolavirus NP gene sequence or an Ebolavirus VP24 gene sequence. Insome embodiments, the heterologous RNA sequence does not encode afunctional Ebola protein (e.g., the heterologous RNA sequence encodestruncated Ebola proteins, Ebola proteins with frame-shift mutationsand/or Ebola protein sequences lacking a start codon). In someembodiments, the heterologous RNA sequence comprises a sequence at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to SEQ ID NO: 2 or SEQ ID NO: 3.

The Sindbis control viruses described herein can be generated using anymethod known in the art. An exemplary method of generating the Sindbiscontrol viruses described herein is illustrated in FIG. 3. In thisexemplary method, capped in vitro transcripts of recombinant RNA bearingsequence of interest and the helper RNA are first synthesized. Thesynthesized RNAs are then electroporated into an appropriate cell, suchas a BHK cell. The Sindbis structural proteins are expressed, but sincethe RNA does not encode replicase enzymes, so no new RNA is transcribed.Recombinant RNA is packaged by the capsid proteins. Viral glycoproteinsassociate with the nucleocapsid and viral particles bud into the culturemedium. The culture supernatant is then collected, filtered and heatinactivated. The viral titer can then be determined using an appropriatemethod, such as real-time PCR and appropriate quality control tests canbe performed to ensure that the RNA is fully encapsulated and there isno contaminating template DNA.

Use of Sindbis Control Vectors in Nucleic Acid Diagnostic Assays

In certain aspects, provided herein are methods of testing a diagnosticassay by running the diagnostic assay on a composition comprising thereplication deficient Sindbis virus described herein. In someembodiments, the diagnostic assay is an assay for the detection ofEbolavirus, an influenza virus, a SARS virus, a hepatitis C virus, aWest Nile virus, a Zika virus, a poliovirus, a measles virus, an HIV-1virus, an HIV-2 virus, an HTLV-I virus and/or an HTLV-II virus. Incertain embodiments, the heterologous RNA sequence in the RNA genome ofthe replication deficient Sindbis virus contains the target sequencedetected in the diagnostic assay.

In some embodiments, the diagnostic assay is a nucleic acidamplification based diagnostic assay. In some embodiments, the nucleicacid amplification based diagnostic assay includes a sample lysis step,a nucleic acid extraction step (e.g., a magnetic-bead based nucleic acidextraction step), a nucleic acid amplification step and/or a nucleicacid detection step. In some embodiments, the nucleic acid amplificationand detection steps are performed simultaneously (e.g., through the useof a real-time detection technology, such as TaqMan probes or molecularbeacons). Examples of nucleic acid amplification processes include, butare not limited to, polymerase chain reaction (PCR), LATE-PCR anon-symmetric PCR method of amplification, ligase chain reaction (LCR),strand displacement amplification (SDA), transcription mediatedamplification (TMA), self-sustained sequence replication (3 SR), Qβreplicase based amplification, nucleic acid sequence-based amplification(NASBA), repair chain reaction (RCR), boomerang DNA amplification (BDA)and/or rolling circle amplification (RCA).

In some embodiments, the diagnostic assay is a nucleic acid sequencingbased diagnostic assay (e.g., a next-generation sequencing baseddiagnostic assay). In some embodiments, the nucleic acid sequencingbased diagnostic assay includes a sample lysis step, a nucleic acidextraction step (e.g., a magnetic-bead based nucleic acid extractionstep), a nucleic acid amplification step, and/or a nucleic acidsequencing step. Examples of nucleic acid sequencing processes include,but are not limited to chain termination sequencing, sequencing byligation, sequencing by synthesis, pyrosequencing, ion semiconductorsequencing, single-molecule real-time sequencing, 454 sequencing, and/orDilute-‘N’-Go sequencing.

EXAMPLES Example 1 Production of an Ebola Sindbis Control Virus

Ebola is a Filovirus with a single stranded, negative sense RNA genome.The Ebola virus genome includes the glycoprotein gene (GP) and thenucleoprotein gene (NP); these two genes were the targets of commonnucleic acid-based diagnostic assays.

Ebola Sindbis Control virus was generated to serve as a control in suchdiagnostic assays. Recombinant Sindbis constructs were designed bycloning either about 2 kb of Ebola Zaire GP gene sequence (SEQ ID NO: 9)or about 1.5 kb of NP gene sequence and about 0.5 kb of a third Ebolagene, VP24 (SEQ ID NO: 10) into the Xba I restriction site of a SinRepSC vector (SEQ ID NO: 1). To ensure that no functional Ebola proteinswould be produced, the constructs were designed to encode severelytruncated GP and NP gene sequences. The GP constructs also lacked theAUG start codon for translation initiation and the NP constructcontained a large internal deletion that changes the reading frame.Engineered stop codons were introduced in both constructs. Thesemeasures increase the safety of the product, but do not interfere withtarget detection (primer and probe binding) of the targeted diagnosticassays.

SEQ ID NO: 9 is an exemplary complete GP Ebola Sindbis control virusgenome. Nucleotides 1 to 7652 and 9708 to 10080 of SEQ ID NO: 9 areSindbis gene sequences, and nucleotides 7653 to 9707 of SEQ ID NO: 9 areEbola GP insert sequences. SEQ ID NO: 10 is an exemplary completeNP/VP24 Ebola Sindbis control virus genome. Nucleotides 1 to 7652 and9976 to 10348 of SEQ ID NO: 10 are Sindbis gene sequences, andnucleotides 7653 to 9975 of SEQ ID NO: 10 are Ebola NP/VP24 insertsequences.

Capped Ebola Sindbis control virus RNA was transcribed in vitro alongwith the helper RNA and introduced into baby hamster kidney cells. At 24hours post-transfection, the cell supernatant was collected and theviral particles were purified and concentrated. Heat treatment wasperformed using a time and temperature known to inactivate similar RNAviruses as a further safety precaution. After titering the viruses usinga TaqMan reverse transcription PCR assay, the viruses were combined anddiluted into defibrinated human plasma containing human genomic DNA and0.09% sodium azide as a preservative.

Three independent lots of the Ebola Sindbis control virus were tested ina real-time nucleic acid amplification based diagnostic assay developedfor the detection of Ebola Zaire virus. The control material wasprocessed identically to how an unknown patient sample would beprocessed. Representative results of this assay are shown in Table 1. Inthis table, Ct is the Cycle threshold value and SAC is the sampleadequacy control (which verifies human source DNA in the sample).

TABLE 1 Ebola Sindbis control virus tested in a Ebola detectiondiagnostic assay. Sample Input SAC ID Volume Test Result GP Ct NP Ct CtLot 1 250 μL Ebola GP DETECTED; 29.5 28.5 35.0 Ebola NP DETECTED Lot 2250 μL Ebola GP DETECTED; 30.6 29.5 34.9 Ebola NP DETECTED Lot 3 250 μLEbola GP DETECTED; 30.3 29.5 34.4 Ebola NP DETECTED

Example 2 Stability of an Ebola Sindbis Control Virus

Stability of quality control materials is critical, especiallyconsidering that for many automated systems, reagents are loaded ontothe instrument and must be stable at ambient temperatures for extendedperiods. Thus, the stability of the Ebola Sindbis Control virus producedas described in Example lunder various storage conditions was tested.

Vials of the Ebola Sindbis Control virus produced as described inExample 1 were subjected to 37° C. At designated time points, vials wereremoved from the stress condition and extracted using the Qiagen QIAampViral RNA Mini Kit. Testing was performed via a TaqMan quantitative realtime PCR assay. Results are shown in FIG. 4 and indicate no loss ofstability after 22 days at 37° C. Using a model based on the Arrheniusequation, this stability at 37° C. correlates with a stability at astorage temperature of 2-8° C. of at least a 2 years.

Vials of the Ebola Sindbis Control virus produced as described inExample lwere subjected to multiple rounds of freezing and thawing(F/T). As shown in FIG. 5, subjecting the Sindbis control virus to threefreeze-thaw cycles did not have an adverse effect on the stability ofthe virus.

To test the extended stability of a Sindbis control vector at varioustemperatures, a recombinant Sindbis virus (bearing 0.8 Kb of targetsequence) was diluted into defibrinated human plasma at 5×10⁵ copies/mLtarget concentration. The material was dispensed into vials and vialswere stored frozen at −20° C., refrigerated at 2-8° C. or at ambient labtemperature (approximately 25° C.) for up to 200 days. Vials were testedperiodically using a TaqMan real time PCR test. No loss of stability wasdetected across the seven months of storage, even for samples stored atambient temperatures. This demonstrates that the viral coat proteins andenvelop of the Sindbis virus form a stable protective barrier thatprevents nucleases in complex clinical matrices such as plasma fromdegrading the target RNA sequence.

Example 3 Production of HIV-1 Multiplex Drug Resistance Sindbis ControlVirus

A Sindbis control virus was generated for use in diagnostic assays forthe detection of drug resistant HIV-1 viruses. The Los Alamos NationalLaboratory HIV Sequence Database was used to generate a “referencesequence” for the control virus. Based on this database as well as thepublication Special Contribution Update of the Drug Resistance Mutationsin HIV-1: March 2013 by Victoria A. Johnson et al., in Topics inAntiviral Medicine, mutations in the HIV-1 genome that confer resistanceto which therapeutic drugs were identified. These mutations and drugsare summarized in Table 2.

TABLE 2 Drug resistant mutations of HIV included in the HIV-1 multiplexdrug resistance Sindbis control virus. Resistance DNA Sequence changeDrug Class Therapy Mutations from reference sequence Protease InhibitorsAtazanavir +/− ritonavir L24I L24I (TTA to ATA) Darunavir/ritonavir D30ND30N (GAT to AAT) Fosamprenavir/ritonavir V32I V32I (GTA to ATA)Indinavir/ritonavir M46I M46I (ATG to ATA) Lopinavir/ritonavir I47V I47V(ATA to CTA) Nelfinavir G48V G48V (GGG to GTG) Saquinavir/ritonavir I50VI50V (ATT to GTT) Tipranavir/ritonavir I54M I54M (ATC to ATG) G73S G73S(GGT to GCT) L76V L76V (TTA to GTA) V82A V82A (GTC to GCC) I84V I84V(ATA to GTA) N88D N88D (AAT to GAT) L90M L90M (TTG to ATG Nucleoside andAbacavir M41L M41L (ATG to TTG) Nucleotide Analogue Didanosine K65R K65R(AAA to AGA) Reverse Emtricitabine D67N D67N (GAC to AAC) TranscriptaseLamivudine T69S insert SS T69S (ACT to TCT and Inhibitors (NRTI)Stavudine insertion of TCC TCC) Tenofovir K70R K70R (AAA to AGA)Zidovudine L74V L74V (TTA to GTA) F77L F77L (TTC to CTC) Y115F Y115F(TAT to TTT) F116Y F116Y (TTT to TAT) Q151M Q151M (CAG to ATG)M184VL210W M184V (ATG to GTG) L210W (TTG to TGG) T215Y T215Y (ACC toTAC) K219Q) K219Q (AAA to CAA) Non-Nucleoside Efavirenz L100I L100I (TTAto ATA) Analogue Reverse Etravirine K101E K101E (AAA to GAA)Transcriptase Nevirapine K103N K103N (AAA to AAC) Inhibitors (NNRTI)Rilpivirine V106A V106A (GTA to GCA) V108I V108I (GTA to ATA) Y181CY181C (TAT to TGT) Y188L Y188L (TAT to TTA) G190A G190A (GGA to GCA)P225H P225H (CCT to CAT) M230L M230L (ATG to CTG)

In addition to the mutations described above, virus entry inhibitordrugs such as Miraviroc are blocked by mutations in the envelop gene.This drug is a CC chemokine receptor 5 (CCR5) antagonists and is onlyeffective for patients with virus that uses the CCR5 co-receptor forviral entry. Viruses that use both CCR5 and CXC chemokine receptor 4(CXCR4) or only CXCR4 will not respond to treatment with CCR5antagonists. A virus's ability to use CXCR4 co-receptor is not definedby a single mutation, but instead is determined by the sequence ofseveral variable “loops” in the gp120 envelop gene.

HXB2 strain of HIV-1 is a CXCR4 utilizing virus. HXB2 sequence isavailable from the Los Alamos National Laboratory HIV Sequence Database.Its sequence was used in the development of the recombinant virusrepresenting the mutant CXCR4 virus. BaL strain of HIV-1 usesexclusively CCR5 co-receptor. Its sequence was obtained from the NCIdatabase and used in the development of recombinant Sindbis virusrepresenting wild type CCR5 virus.

Four DNA sequences were chemically synthesized and cloned into the Xba Irestriction site of a SinRep SC Sindbis expression plasmid (SEQ ID NO:1), which bears genes required for Sindbis virus production. FourSindbis control viruses were generated, one that contained the 5′ end ofa wild-type HIV-1 genome, one that contained the 5′ end of a multidrugresistant HIV-1 viral genome, one that contained the 3′ end of awild-type HIV-1 genome and one that contained the 3′ end of a multidrugresistant HIV-1 viral genome. The insert sequences for these fourcontrol viruses are described in Table 3.

TABLE 3 Description of recombinant virus sequences. Genes included inthe HXB2-Nucleotide Construct Designation sequence Positions 5′multi-mutant (SEQ ID part of p7, p1, p6, Protease, Contains continuousNO: 11) RT, p51 RNAse and sequence from nucleotides Integrase 1900through 5400. The mutations shown in Table 1 are incorporated 5′ WT (SEQID NO: 12) part of p7, p1, p6, Protease, Contains continuous RT, p51RNAse and sequence from nucleotides Integrase 1900 through 5400. 3′mutant (SEQ ID NO: 13) A portion of gp120 nucleotides 6300-7825 ofincluding V1-V5 variable HXB2 sequence are loops included 3′ WT (SEQ IDNO: 14) A portion of gp120 The BaL sequence which including V1-V5variable corresponds to HXB2 6300-7825 loops (as determined by BLASTalignment) is included

SEQ ID NO: 11 is the DNA counterpart to an exemplary complete 5′multi-mutant HIV-1 Sindbis control virus genome. Nucleotides 1 to 7646and 11167 to 11655 indicate Sindbis gene sequences, and nucleotides 7647to 11166 indicate multi-mutant HIV-1 insert sequences. SEQ ID NO: 12 isthe DNA counterpart to an exemplary complete 5′ wild-type HIV-1 Sindbiscontrol virus genome. Nucleotides 1 to 7646 and 11161 to 11649 indicateSindbis gene sequences, and nucleotides 7647 to 11160 indicate wild-typeHIV-1 insert sequences. SEQ ID NO: 13 is the DNA counterpart to anexemplary complete 3′ mutant HIV-1 Sindbis control virus genome.Nucleotides 1 to 7646 and 9187 to 9675 indicate Sindbis gene sequences,and nucleotides 7647 to 9186 indicate mutant HIV-1 insert sequences. SEQID NO: 14 is the DNA counterpart to an exemplary complete 3′ wild-typeHIV-1 Sindbis control virus genome. Nucleotides 1 to 7646 and 9182 to9670 indicate Sindbis gene sequences, and nucleotides 7647 to 9181indicate wild-type HIV-1 insert sequences.

The process used to produce the recombinant HIV-1 Sindbis controlviruses is outlined in FIG. 7. Briefly plasmids that contain the targetHIV-1 sequences in the SinRep vector were linearized with Not Irestriction enzyme. An aliquot was analyzed by agarose gelelectrophoresis to ensure that DNA cutting is complete.

Ambion mMessage mMachine SP6 kit was used for in vitro transcription oflarge amounts of capped RNA using reaction conditions optimized for longtranscripts. DHBB is a helper RNA needed for packaging of thereplication defective Sindbis virus; this helper RNA was transcribedfrom a linearized plasmid as well. The integrity and identity of thetranscribed RNA was analyzed by denaturing agarose gel electrophoresis.The RNA was treated with DNAse to remove template plasmid DNA andpurified using Ambion MegaClear kit.

To ensure optimal cell viability, BHK-21 (Baby Hamster Kidney cells)were amplified in culture for 2-4 passages after revival of frozenstock. Immediately prior to electroporation, the fetal bovine serum inthe culture media was reduced, which helps reduce this cell's tendencyto form clumps. Preventing cell clumps is desirable to maximize thetransfection efficiency during electroporation.

The in vitro transcribed RNA was introduced into the BHK-21 cells viaelectroporation. The cells were washed at 6 hours post transfection toremove any unincorporated RNA.

The in vitro transcribed RNAs (HIV-1 sequences in SinRep RNA and DHBBhelper RNA) were translated within the cells to produce the proteinsrequired for recombinant Sindbis virus assembly and budding. Therecombinant viruses were released into the culture media. The culturemedia was collected at 24 hours post transfection. The crude viralsupernatant and the purified viruses were titered by extracting theviral nucleic acids using the Qiagen QlAamp Viral RNA mini kit and thenusing quantitative TaqMan real time PCR assay which targets a portion ofthe Sindbis viral vector RNA.

Example 4 Production of a Zika Sindbis Control Virus

Zika virus is a positive-sense, single-stranded RNA molecule of about10794 bases long, and it codes a single polyprotein that is subsequentlycleaved into capsid (C), precursor membrane (prM), envelope (E), andnon-structural proteins (NS). Zika virus reference materials weredesigned based on a 2007 Zika virus strain with GenBank Accession numberEU545988.1 (SEQ ID NO: 15). For the Zika Reference Materials, thisgenome was divided across four different constructs with at least ˜150bp overlap between constructs and breakpoints at the ends of conserveddomains. The overlap design is shown in FIGS. 9 and 10.

There was a 152 bp overlap between the “Zika Env” and “Zika NS2/NS3”construct, 150 bp overlap between “Zika NS2/NS3” and “Zika N54”construct and 180 bp overlap between “Zika N54” and “Zika NS5”constructs. These overlaps are designed to cover any diagnostic assaysthat target the ends of conserved domains. All four constructs weresynthesized and introduced into Sindbis plasmids, which were used toprepare recombinant Sindbis virus.

The recombinant Zika/Sindbis virus were expressed, and high titer stocksolutions of the viruses were prepared. The high titer stock solutionsof recombinant Zika/Sindbis virus were diluted 1:100 in PBS, and RNA wasextracted and eluted into 120 μL of 1:10 diluted AVE buffer. ExtractedRNA was assayed by droplet digital PCR using a one-step RT-ddPCR mastermix (Bio-Rad, 186-4021) at neat and 1:10 dilutions. Vector specificprimer/probe sets were used for quantifying all four constructs as shownin Table 4.

TABLE 4 Quantification of Zika Construct Copy Numbers in Zika VirusReference Materials Average Copies Copies per copies per Copies per BackPer μL of μL mL of calculated 20 μL Extracted Extracted Extracted copiesper Sample Well RNA RNA Sample mL stock Zika Env 3580 716 6.81E+025.84E+05 5.84E+07 Zika Env 2800 560 Zika Env 3840 768 Zika Env 1:10 39679.2 7.84E+01 6.72E+04 6.72E+07 Zika Env 1:10 388 77.6 Zika Env 1:10 39278.4 Zika NS2/NS3 6040 1208 1.20E+03 1.03E+06 1.03E+08 Zika NS2/NS3 63601272 Zika NS2/NS3 5580 1116 Zika NS2/NS3 692 138.4 1.27E+02 1.09E+051.09E+08 1:10 Zika NS2/NS3 636 127.2 1:10 Zika NS2/NS3 578 115.6 1:10Zika NS4 6460 1292 1.29E+03 1.10E+06 1.10E+08 Zika NS4 5380 1076 ZikaNS4 7480 1496 Zika NS4 1:10 532 106.4 1.33E+02 1.14E+05 1.14E+08 ZikaNS4 1:10 750 150 Zika NS4 1:10 718 143.6 Zika NS5 2528 505.6 5.33E+024.57E+05 4.57E+07 Zika NS5 2268 453.6 Zika NS5 3200 640 Zika NS5 1:10264 52.8 5.13E+01 4.40E+04 4.40E+07 Zika NS5 1:10 196 39.2 Zika NS5 1:10310 62

Based on the high titer stock concentration, a 35 mL bulk was formulatedat 5.0E+05 copies/mL in filtered human plasma (Basematrix) containing0.09% NaN₃ diluent and human genomic DNA (H9 DNA, 50 ng/mL). A PallAcropak 1000 Filter Capsule (PES RM-1002220) was used for filtering theplasma. To 900 mL of filtered plasma, 810 mg of sodium azide and 45 μgof human genomic DNA was added and mixed for 15 minutes. All fourconstructs were targeted to 5.0E+05copies/mL in the prepared bulk. Bulkwas mixed thoroughly for about 15 minutes, and RNA was extracted intriplicate and assayed using ddPCR with a One-Step RT-PCR master mixfrom Bio-Rad Laboratories (Catalogue# 186-4021). Assay specificprimers/probe were used to quantify each construct. Data is shown inTable 5.

TABLE 5 Quantification of Zika Construct Copy Numbers in Zika VirusReference Materials Formulated with Human Plasma Copies Copies per perμL of Average 20 μL Extracted Copies per Copies per Sample Conc. WellRNA mL of bulk mL of bulk Zika Env 50.8 1016 203.2 1.74E+05 1.72E+05Zika Env 48.6 972 194.4 1.67E+05 Zika Env 50.7 1014 202.8 1.74E+05 ZikaNS2/NS3 33.6 672 134.4 1.15E+05 1.22E+05 Zika NS2/NS3 37.3 746 149.21.28E+05 Zika NS2/NS3 36.2 724 144.8 1.24E+05 Zika NS4 125.6 2512 502.44.31E+05 4.07E+05 Zika NS4 117.2 2344 468.8 4.02E+05 Zika NS4 113.1 2262452.4 3.88E+05 Zika NS5 207 4140 828 7.10E+05 6.88E+05 Zika NS5 197 3940788 6.75E+05 Zika NS5 198 3960 792 6.79E+05

An Altona Realstar Zika RT-PCR assay was performed on the extracted RNAfrom prepared bulk. The Altona Zika RT-PCR assay is a qualitative assaythat gives a Positive or Negative result as shown in Table 6. Data isshown for both Zika and internal control analytes. The internal control(IC Zika (JOE)) should be detected in all negative and positive wellsfor a valid result, whereas Zika signal (Zika (FAM)) should be detectedonly in Positive wells. Bulk was tested in five replicates with Ctvalues around 28. Negative control was undetermined as expected, and thepositive control Ct was 32.

TABLE 6 Altona Realstar Zika RT-PCR Assay Performed on Zika VirusReference Materials Formulated with Human Plasma Well Sample NameDetector Task Ct Result A1 Zika Bulk Zika (FAM) Unknown 28.4665 POSITIVEA1 Zika Bulk IC Zika (JOE) Unknown 30.7525 VALID A2 Zika Bulk Zika (FAM)Unknown 28.5619 POSITIVE A2 Zika Bulk IC Zika (JOE) Unknown 30.9062VALID A3 Zika Bulk Zika (FAM) Unknown 28.5517 POSITIVE A3 Zika Bulk ICZika (JOE) Unknown 30.9627 VALID A4 Zika Bulk Zika (FAM) Unknown 28.7069POSITIVE A4 Zika Bulk IC Zika (JOE) Unknown 31.0635 VALID A5 Zika BulkZika (FAM) Unknown 28.6494 POSITIVE A5 Zika Bulk IC Zika (JOE) Unknown31.0911 VALID C1 Negative control Zika (FAM) NTC Undetermined NEGATIVEC1 Negative control IC Zika (JOE) NTC 30.8181 VALID C2 Positive ControlZika (FAM) Unknown 32.0931 POSITIVE C2 Positive Control IC Zika (JOE)Unknown 30.809  VALID

6 mL of prepared bulk was sent to a commercial laboratory for bioburdentesting. The bioburden result was 0 cfu/mL for bacterial growth and theZika reference materials passed the acceptance criteria (<100cfu/mL orNo growth).

Extracted viral RNA from recombinant Sindbis virus was sequence-verifiedby Sanger sequencing. All four constructs were PCR amplified at thebeginning and end of the insert, and each nucleotide sequence displayed100% sequence homology with the EU545988.1 sequence used to design theconstructs (SEQ ID NO:15).

Example 5 Stability of an Influenza Sindbis Control Virus

An influenza reference material comprising an 800-nucleotide sequence ofthe H7N9 influenza virus was constructed using methods similar to thosedescribed above. The influenza reference material was diluted intoaqueous buffer or defibrinated human plasma at 5×10⁵ copies/mL in acommutable matrix. The material was dispensed into vials and stored at−20° C., 4° C., or room temperature (˜25° C.). Vials were testedperiodically using a laboratory developed H7N9 TaqMan real time PCRtest. As shown in FIGS. 11 and 12, the influenza reference materialstored at ambient temperature for 500 days was stable as only ˜15% lossof signal was observed. This stability profile suggests that the viralcoat and envelope proteins form a stable protective barrier thatprevents nucleases in complex clinical matrices (such as plasma) fromdegrading the target RNA sequence.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A replication deficient recombinant Sindbis virus comprising a RNAgenome comprising: an open reading frame (ORF) encoding functionalSindbis non-structural proteins; and a heterologous RNA sequence.
 2. Thereplication deficient recombinant Sindbis virus of claim 1, wherein theORF encoding functional Sindbis non-structural proteins is located 5′ ofthe heterologous RNA sequence.
 3. The replication deficient recombinantSindbis virus of claim 1, wherein the ORF encoding Sindbisnon-structural proteins has a nucleotide sequence that is at least 90%identical to nucleotides 1-7648 of SEQ ID NO:
 1. 4. (canceled)
 5. Thereplication deficient recombinant Sindbis virus of claim 1, wherein theRNA genome lacks a sequence encoding a functional version of one or moreof the Sindbis structural proteins. 6.-9. (canceled)
 10. The replicationdeficient recombinant Sindbis virus of claim 1, wherein the heterologousRNA sequence comprises a non-Sindbis RNA virus sequence or a retrovirussequence. 11.-16. (canceled)
 17. The replication deficient recombinantSindbis virus of claim 10, wherein the heterologous RNA sequencecomprises a non-Sindbis RNA virus sequence.
 18. The replicationdeficient recombinant Sindbis virus of claim 17, wherein the non-SindbisRNA virus sequence is an Ebolavirus sequence, an influenza virussequence, a SARS virus sequence, a hepatitis C virus sequence, a WestNile virus sequence, a Zika virus sequence, a poliovirus sequence, or ameasles virus sequence.
 19. The replication deficient recombinantSindbis virus of claim 18, wherein the non-Sindbis RNA virus sequence isan Ebolavirus sequence. 20.-26. (canceled)
 27. The replication deficientrecombinant Sindbis virus of claim 10, wherein the heterologous RNAsequence comprises a retrovirus sequence.
 28. The replication deficientrecombinant Sindbis virus of claim 27, wherein the retrovirus sequenceis an HIV-1 sequence, an HIV-2 sequence, an HTLV-1 sequence, or anHTLV-II sequence.
 29. The replication deficient recombinant Sindbisvirus of claim 28, wherein the retrovirus sequence is an HIV-1 sequence.30.-65. (canceled)
 66. A composition, comprising a replication deficientSindbis virus of claim
 1. 67. The composition of claim 66, wherein thereplication deficient Sindbis virus comprising a RNA genome comprising asequence that is at least 90% identical to SEQ ID NO: 11; and areplication deficient Sindbis virus comprising a RNA genome comprising asequence that is at least 90% identical to SEQ ID NO:
 13. 68. (canceled)69. The composition of claim 66, wherein the replication deficientSindbis virus comprising a RNA genome comprising a sequence that is atleast 90% identical to SEQ ID NO: 12; and a replication deficientSindbis virus comprising a RNA genome comprising a sequence that is atleast 90% identical to SEQ ID NO:
 14. 70. (canceled)
 71. The compositionof claim 66, wherein the replication deficient Sindbis virus comprisinga RNA genome comprising a sequence that is at least 90% identical toeither nucleotides 1-3446 of SEQ ID NO: 15, nucleotides 3294-5575 of SEQID NO: 15, nucleotides 5425-7722 of SEQ ID NO: 15, or nucleotides7542-10272 of SEQ ID NO:
 15. 72.-77. (canceled)
 78. A nucleic acidmolecule encoding the RNA genome of the replication deficient Sindbisvirus of claim
 1. 79. The nucleic acid molecule of claim 78, wherein thenucleic acid molecule is an RNA molecule. 80.-82. (canceled)
 83. Amethod of making a replication deficient Sindbis virus comprising: (a)transfecting a cell with the RNA molecule of claim 79 encoding the RNAgenome of the replication deficient Sindbis virus and a helper RNAmolecule encoding functional Sindbis structural proteins; (b) culturingthe transfected cell of step (a) under conditions such that the cellproduces a replication deficient Sindbis virus comprising the mRNAgenome; and (c) collecting the replication deficient Sindbis virus. 84.(canceled)
 85. A method of testing a diagnostic assay, comprisingperforming the diagnostic assay on a composition of claim 66.